A wide variety of animals are able to detect the Earth’s magnetic field and use it as a source of directional information2,3
. In many cases, animal magnetic detection appears to depend on chemical reactions initiated by specific wavelengths of light4,5
. The most popular chemical reaction model proposes that light-dependent magnetoreception is mediated by radical pair reactions that are generated in specialized photoreceptors6,7,8
Because of their photoreceptor function and biochemical properties, the DNA photolyase-related Crys have been postulated to be the key photoreceptor molecules that generate magnetically sensitive radical-pair products7,9
. However, all animals Crys are not functionally equivalent. In fact, two phylogenetically and functionally distinct groups of animal Crys have been identified and characterized based largely on their roles in the regulation of circadian clocks10,11
-like Type 1 Crys are sensitive to ultraviolet-A/blue wavelengths of light12,13
and function mainly as circadian photoreceptors. Vertebrate-like Type 2 Crys, on the other hand, are thought to function primarily as negative regulators of the clock’s transcriptional feedback loop, the quintessential intracellular gear of the molecular clock. Although both types of Cry appear to be widespread in the animal kingdom, there is considerable variation in their distribution among taxa11
; insects can have Type 1 (the only one found in Drosophila
), Type 2, or both types of Cry, whereas vertebrates only have Type 2 Crys. Type 2 Cry proteins are thought to mediate light-dependent magnetoreception in many vertebrate groups14
including migratory birds, but direct evidence for such a role has yet to be established for any animal due to the lack of available tools for genetic manipulation.
Recently, we integrated genetic and behavioural approaches to show that light-dependent magnetoreception in Drosophila
is mediated by its Type 1 Cry1
. The necessity of Drosophilia
Cry for magnetosensitivity was shown using two distinct Cry-deficient mutations, which abolished magnetosensitive behavioural responses. This work established Drosophila
as a viable genetic system to delineate the molecular underpinnings of Cry-based magnetoreception in animals.
We began our current investigations by showing that a Drosophila cry
transgene can rescue magnetosensitivity in Cry-deficient flies. We used the GAL4-UAS system, with timeless
as the driver, which drives transgene expression in tim
-expressing and most cry
. We attempted rescue of magnetosensitive behavioural responses in the Cry loss-of-funtion cryb
mutant background by expressing a UAS- Drosophila cry
transgene. Magnetosensitivity was assessed using our behavioural assay in which flies experience an electric coil-generated magnetic field and display their behavioural responses in a binary-choice T-maze housed in an illuminated black box1
. The two-coil system produces a magnetic field on one side of the T-maze, while producing no field on the opposite side. Flies were tested for either their response to the magnetic field in the naïve state or after a training session in which the field was paired with a sucrose reward. Response to the magnetic field was expressed as a Preference Index (PI), with negative values representing an avoidance of the field and positive values a preference for the field. As previously described, wild-type Canton-S flies expressing endogenous Cry show differential behavioural responses to the magnet in the T-maze – a naïve avoidance of the field and a trained preference for the field (white and black bars, respectively; , left bar set)1
. This establishes a standard of behavioural responses for the transgenic studies.
Type 1 Crys rescue light-dependent magnetoreception in Cry-deficient flies
Predictably, Cry-deficient cryb lines expressing either the tim-GAL4 driver alone or the Drosophila cry transgene alone did not respond to the magnetic field (, middle two bars sets). However, cryb flies expressing the Drosophila cry transgene under control of the tim-GAL4 driver now manifested significant naïve and trained responses to the field (tim-GAL4/UAS-dcry; , right bar set), similar in both direction and magnitude to those of wild-type flies (, left bar set). Thus, the tim-GAL4 driver expresses Cry appropriately so that it can rescue the magnetosensitive behavioural responses in the cryb background, making possible study of the biochemical mechanism through which animal Crys mediate magnetosensitivity.
We next used the tim
driver in cryb
flies for evaluating the functional roles of monarch butterfly Crys in magnetosensitivity. Monarchs have both a Type 1 Cry (designated Cry1) and a Type 2 Cry (designated Cry2). In vitro
and in vivo
studies have shown that they are functionally distinct16
; Cry1 functions as a circadian photoreceptor, while Cry2 functions as a core clock component. Likewise, monarch Cry2 does not exhibit light sensitivity, at least in the assay systems studied so far10,11
. Monarch butterfly Crys thus provide a unique opportunity to directly compare the functional equivalency for magnetosensitivity of Type 1 and Type 2 Crys from the same species in vivo
, using transgenesis in Drosophila
A monarch cry1
transgene driven by tim
-GAL4 successfully restored both naïve and trained responses to the magnetic field in the cryb
; ). Furthermore, we showed that monarch Cry1 rescues magnetosensitivity in a wavelength-dependent manner ( and ). Similar to Drosophila
, monarch Cry1-rescued flies exhibited significant naïve and trained responses to a magnetic field under full-spectrum light (~300–700 nm) and under UV-A/blue light (<420 nm), but the behavioural responses were abolished when only long wavelength light (>420 nm) was available. Thus, at the intensities used, light in the UV-A/blue range is both necessary and sufficient for monarch Cry1 to function in the Drosophila
magnetoreception system. Together, these results indicate that Type 1 Crys play a key role in the magnetic transduction pathway of Drosophila
and provide evidence for the functional equivalency of Type 1 Crys in magnetoreception across insect species.
Remarkably, a monarch cry2
transgene driven by tim
also successfully restored both naïve and trained responses to the magnet in the cryb
; ) and did so in a wavelength-dependent manner similar to that of the monarch cry1
transgene (). The reliance of monarch Cry2-induced magnetosensitivity on UV-A/blue light is consistent with the predicted spectral sensitivities of chicken Type 2 Crys, as measured from the embryonic iris ex vivo17
. To our knowledge, these are the first genetic data to show that a Type 2 Cry can function in a light-dependent behavioural response; another transgenic study has suggested a light-induced proteolytic response for a Type 2 protein18
. The light-dependence of magnetosensitivity in flies expressing monarch cry1
transgenes suggests that both proteins undergo the photochemical reactions necessary for magnetic sensing.
Monarch Type 2 Cry rescues light-dependent magnetosensivitiy in Cry-deficient flies
The widely held radical pair model for Cry-mediated magnetoreception is derived from the biochemical reactions proposed for the photocycle of plant Crys6
. In this model, magnetically sensitive radical pair products are generated when the fully oxidized flavin adenine dinucleotide (FAD) chromophore is photoreduced through a sequence of intraprotein electron transfers along a chain of three tryptophan residues; these three residues are conserved in photolyases and Crys (the so-called ‘trp triad’; Supplementary Fig. S1
. It is well established that blue light in the 450 nm range is sufficient for the photoreduction of oxidized flavin and the generation of trp triad-mediated radicals 6,12,18,19,20
. The lack of a magnetic response for both Type 1 and Type 2 Crys under wavelengths >420 nm suggests that neither type signals through the photoreduction of oxidized flavin. The results further imply that the photoactivation of either Cry type for magneto-sensing does not depend on the presence of trp triad-mediated radical pairs.
We directly tested whether the trp triad pathway is necessary for Cry-mediated magnetoreception by mutating the terminal tryptophan (W) to phenylalanine (F) in Drosophila
Cry (W342F, two independent UAS
lines) as well as in monarch Cry1 (W328F, three independent lines) and in monarch Cry2 (W345F, two independent lines). Magnetosensitivity was then assessed in cryb
flies expressing each of the trp-mutated Crys as UAS
transgenes under control of the tim
driver. These mutations were chosen because previous biochemical studies have shown that mutating this tryptophan in an identical way in Drosophila
Cry and in monarch Cry1 blocks photoreduction of oxidized FAD in vitro12,20,
thus preventing the generation of trp triad-mediated radical pair products. No such biochemical data exist for any Type 2 Cry protein.
Mutating the terminal tryptophan in Drosophila
Cry and monarch Cry1 did not affect the ability of transgenic flies to respond to the magnetic field, as lines of both tim-GAL4/UAS-dcry
W342F and tim-GAL4/UAS-dpcry1
W328F flies exhibited appropriate, significant naïve and trained responses in the cryb
background ( and ), as expected based on the lack of magnetic sensing under >420 nm light. Notably, the trp triad is important but not essential for Drosophila
Cry to function in the circadian system, because the trp mutant trangenes modestly rescued circadian photoreceptive responses in cryb
flies (Supplementary Fig. S2
). Although other ways of generating radical pairs in Cry have been proposed, for example through superoxides, all described radical pair mechanisms depend on a functional trp triad6,21,22
. Therefore, radical pairs generated through those mechanisms are not necessary for magnetoreception through Type 1 Crys.
Effects of terminal tryptophan mutations on Type 1 and Type 2 Cry-mediated magnetosensitivity
Another possibility is that there are light-sensitive biochemical reactions for magnetosensing that involve Type 1 Crys but do not
require a functional trp triad, such as those that may occur in a photolyase-like photocycle12,20
. In this photocycle, UV-A/blue light (<420 nm) induces reduced flavin to initiate electron transfer to an unknown substrate that generates a radical and initiates the signal; back transfer turns off the signal23
. Whether this photocycle could generate radical pairs appropriate for magnetoreception warrants consideration.
Surprisingly, magnetosensitivity was not restored in either of the two transgenic lines expressing the monarch Cry2 trp mutation (tim-GAL4/UAS-dpcry2
W345F) in the cryb
background (), even though protein expression was stable (Supplemental Fig. S3
). The Cry2 trp mutation did alter the ability of the protein to function as a transcriptional repressor, although the mutant protein could still clearly repress Clock:Cycle-mediated transcription in a dose-dependent manner in cell culture (). Because monarch Cry2 cannot rescue magnetic sensing under >420 nm light (), the lack of a magnetic response in Cry2 trp mutant flies under full-spectrum light is unlikely caused by the absence of radical pairs generated via the trp-triad pathway. Rather, the Cry2 trp mutation may have caused a nonspecific effect on magnetosensing function due to protein misfolding. Thus, Cry2 may use a photochemical mechanism common to both Type 1 and Type 2 Cry, such as the photolyase-like photocycle mentioned above. Alternatively, Cry2 could use a photochemical mechanism that is dependent on the terminal trp, but independent of trp triad-generated radical pairs.
A recent report described a Cry-mediated effect of magnetic fields on circadian period in Drosophila
studied under constant light24
. In the circadian system of Drosophila
, Cry interacts with the key clock protein Timeless (Tim) in a light-dependent manner, leading to its degradation and subsequent effects on circadian period25
. Thus, this reported light-induced magnetosensitive effect of Cry on circadian period is entirely dependent on its interaction with Tim. It is important to note that the behavioural responses to a magnetic field in the present study do not depend on Tim; Tim-deficient tim0
mutant flies showed robust naïve and trained responses to the magnetic field in the T-maze ().
The clock proteins Tim and Cyc are not required for Drosophila magnetoreception
results also show that light-dependent magnetoreception through Type 1 Crys is not mediated through photochemical interactions between Cry and Tim. In addition, other core clock proteins are not likely involved in magnetoreception. Indeed, flies deficient in the essential clockwork transcription factor Cycle (homozygous cyc0
) also showed a strong response to the magnetic field (), and we have shown previously that disrupting the molecular clock under constant light conditions does not affect magnetosensitivity in wild-type Canton-S flies1
Our results advance the field of magnetoreception in animals by strongly suggesting that both Type 1 and Type 2 Crys can function in magnetosensitive responses without generating radical pairs via the trp triad, which is widely thought to be the critical element of a Cry-based magnetoreception mechanism6,7,9
. The future challenge will be understanding more precisely how magnetosensitive chemical reactions are actually generated, and determining how, once generated, they are transduced into neural signals that lead to behavioural responses. It is noteworthy that a recent study suggests that light-activated Drosophila
Cry can directly modulate ion channel activity in fly brain, independent of its light effects on the molecular clock mechanism27
. Although more work is needed to address the mechanistic details of this possibility, it is an exciting way in which magnetosensitive Cry of either type could transduce directional information to the brain for behavioural responses.
Our foray into animal magnetoreception was initiated on the idea that monarch butterflies, like other long-distance migrants, used several navigational strategies, including magnetoreception28
. Indeed, our transgenic results now show that both
types of monarch Cry proteins have the molecular capacity to transduce magnetic information, and the hunt is now on for a light-sensitive behavioural correlate of magnetosensitivity in migrating monarchs. This study highlights how the unique biology of monarch butterflies continues to advance our understanding of general biological principles.